Polyploids are organisms with more than two sets of chromosomes
(Levin, 2002), and though polyploidy is relatively rare in most groups
of animals (Otto and Whitton, 2000; Mable, 2004; but see Legatt and
Iwama, 2003), it is extremely common in flowering plants. Indeed,
studies suggest 70% or more of all angiosperms have experienced one or
more rounds of genome duplication at some point during their
evolutionary history (Grant, 1963; Stebbins, 1971; Goldblatt, 1980;
Masterson, 1994; Otto and Whitton, 2000; Cui et al, 2006). Polyploids
can differ from their diploid relatives in a variety of ways, including
but not limited to cytology, gene activity, physiology, development,
tolerance to pests and/or pathogens, stress tolerance, competitive
ability, and reproductive strategy (Levin, 1983, 2002; Barker et al.,
2016). Despite these differences, relatively few studies have compared
ecological and evolutionary patterns and processes in natural
populations of closely related polyploids and diploids. In particular
relatively little is known about the consequences of genome duplication
in awfopolyploids, despite growing evidence that they are common in
nature (Soltis et al, 2007). Such studies are important because they
help us understand the ecological and evolutionary consequences of
polyploidy without confounding the effects of genome duplication with
interspecific hybridization.

Polyploidy is often thought to lead to "instant
speciation" due to reductions in the viability and fertility of
intercytotype hybrids (Husband and Sahara, 2003; Bolnick and
Fitzpatrick, 2007; Rieseberg and Willis, 2007; Wood et al., 2009;
Ramsey, 2011). This reduction in intercytotype hybrid fitness, also
reflected in the concept of minority cytotype disadvantage (Levin,
1975), is expected to select for increased prezygodc isolation. Such
isolation could occur as a consequence of changes in flowering time
(Lumaret et al., 1987; Thompson and Lumaret, 1992; Ramsey, 2011),
increased levels of self-fertilization (Stebbins, 1950; Barringer, 2007)
and/or changes in the activity and composition of pollinator communities
(Thompson and Merg, 2008; Segraves and Anneberg, 2016). Alternatively,
the ploidy change itself might alter any of these traits; e.g.,
flowering time (Ramsey, 2011) or self-incompatibility systems (Miller
and Venable, 2000). Recent work has found prezygodc isolation is often
larger than post-zygotic isolation, supporting these ideas (Husband et
al., 2016). However, geographic separation is a substantial contributor
to this isolation. We have less information on the reproductive ecology
and its impact on the potential for gene exchange in natural
geographically proximate populations of different cytotypes.

Galax is a natural autopolyploid series that includes diploid,
triploid, and tetraploid individuals occurring in uniform- as well as
mixed-cytotype populations (Baldwin, 1941; Nesom, 1983; Burton and
Husband, 1999; Servick et al., 2015). Genome duplication has occurred
numerous times in the species, and a recent study suggests 46
independent origins of tetraploid Galax, more than for any other
polyploid (Servick et al., 2015). These characteristics bolster the
species' reputation as "the classic example" of
autopolyploidy (Baldwin, 1941; Stebbins, 1950; Grant, 1971; Soltis et
al., 2007). However, despite this reputation, surprisingly little is
known about the basic ecology of the species (but see Johnson et al.,
2003) and virtually nothing is known about its reproductive biology.
Indeed, the mating system of Galax has not been investigated, though
levels of heterozygosity suggest that it is at least partially
outcrossing (Servick et al., 2015). Nearly two-thirds of populations
located in the most dense region of cytotype overlap contain multiple
cytotypes (Burton and Husband, 1999). Therefore, an understanding of the
species' reproductive ecology would be of value, as it would help
us better understand why triploid and tetraploid lineages of Galax have
arisen so frequendy, the overall lack of genetic differences among
cytotypes (Servick et al., 2015) and whether we should expect levels of
genetic differentiation among cytotypes to increase in the future.

We studied six populations (two diploid, two tetraploid, and two of
mixed ploidy; i.e., containing both diploids and tetraploids) of Galax
located in an area where both cytotypes are common. Our goals were to
contribute to understanding of the basic reproductive biology and
ecology of the species and to compare diploids and tetraploids with an
eye toward assessing the potential for intercytotype gene movement. More
specifically, we addressed four questions: (1) Do the flowering
phenologies of diploids and tetraploids differ? (2) Do diploids and
tetraploids differ in terms of the taxonomic compositions or abundances
of their floral visitors? (3) Do levels of self-compatibility differ
between diploids and tetraploids and does the species produce seed
autonomously? (4) Do diploids and tetraploids differ in terms of pollen
limitation and seed production?

Cytotype frequencies are spatially structured throughout the
species' range, with diploids being most common in the north and
tetraploids and rare triploids being most frequent in the south;
however, a substantial region of geographic overlap of the cytotypes
exists, particularly near the Blue Ridge escarpment (Nesom, 1983; Burton
and Husband, 1999; Servick et al., 2015). At finer spatial scales,
diploids and tetraploids are found in similar habitats (Servick et al.,
2015), although some ecological differences between cytotypes do exist
(Nesom, 1983; Johnson et al., 2003). In the region of overlap, almost
two-thirds of populations contain multiple cytotypes (Burton and
Husband, 1999). Populations occur in shady forested areas, and
population sizes range widely, from fewer than 10 to many thousands of
individuals (Baldwin, 1941; Nesom, 1983; Burton and Husband, 1999).

Plants are hermaphroditic with individuals reproducing asexually
via stolons and sexually via small white flowers attached to long
spike-like racemes (Baldwin, 1941; Nesom, 1983; Burton and Husband,
1999). Depending on population location, the species begins to flower in
May and continues into July with the peak of flowering during the month
of June (B. Barringer, pers. obs.). Whether Galax can reproduce via
self-fertilization is not known; however, other members of the family
are self-compatible (Ferrer and Good, 2012).

Sampling.--Six populations were used in this study, including two
comprised solely of diploid individuals, two comprised solely of
tetraploid individuals, and two of mixed-ploidy (i.e., including diploid
and tetraploid individuals) (Table 1). For the analyses reported here
the diploids and tetraploids from these mixed populations were treated
as though they were from separate populations to permit comparing the
performance of diploids and tetraploids. Populations with triploid
individuals were not included in this study.

Assessing ploidy.--Ploidy was assessed and verified for at least 42
haphazardly-selected plants in each population using chromosome squashes
(cf., Baldwin, 1941; Nesom, 1983). Because Galax can reproduce clonally
via stolons, it is sometimes difficult to determine where one plant ends
and another begins. To reduce the likelihood of sampling twice from the
same individual within a study population, we focused on plants
spatially separated from each other by >2 m (often >3 m). If the
first 42 individuals sampled from a given population were found to be of
a single cytotype (diploid or tetraploid), that population was assumed
to be comprised solely of either diploid or tetraploid plants. For
populations of mixed ploidy at least 21 individuals of each cytotype
were identified. In all cases fresh young leaf tissue was harvested from
each individual, fixed in a 3:1 ethanol:acetic acid solution, immersed
in aceto-carmine stain, and boiled five times. Stained tissue was
macerated and squashed on a microscope slide and chromosomes were
visualized with a light microscope using oil-immersion (100X).
Individuals were assessed as being either diploid (2n = 2x = 12) or
tetraploid (2n = 4x = 24) and were used in one of the studies of Galax
reproductive ecology detailed below.

Flowering phenology and number.--The flowering phenologies of six
plants in each population were studied by counting the number of open
flowers on each individual once/week throughout their flowering period
(6 wk total; May 25 through July 11, 2009). In mixed-ploidy populations,
three diploid and three tetraploid plants were used. Open flowers were
defined as those whose petals were at least partially reflexed. Seasonal
patterns of flower production for each plant were summarized by the
"average flower time," representing the census week that the
mean flower was produced. This was calculated by weighting each census
week by the proportion of an individual's flowers that were open
that week. Open flowers were also summed over the flowering period on
each plant to produce an index of total flower production. Average
flower time and total flower production were natural log-transformed and
ANOVA was used to compare populations. Population was treated as a fixed
effect because populations were selected to be diploid, tetraploid or
mixed. A priori contrast was then used to compare diploid populations to
tetraploid populations. Throughout, all means are reported with standard
errors.

Floral visitor observations.--Once flowering began, and continuing
through the majority of the flowering period (6 wk), floral visitor
observations were conducted weekly in each study population by two
observers during 1 h intervals at three different times of the day:
morning (0800-0900), afternoon (1300-1400), and evening (1800-1900), for
a total of 18 observation periods per population. During each 1 h time
interval, each observer visually monitored four to six plants and
recorded the identities of floral visitors (taxonomic order) as well as
the frequencies and lengths of their visits in seconds. The observation
periods were lumped for each population for analysis. Potential
differences among populations and pollinator taxa in the number of
visitors and duration of visits were evaluated using a two-way ANOVA
with a priori contrasts to compare diploid populations to tetraploid
populations. The distributions of both variables met assumptions of
ANOVA.

Self-compatibility and autonomous self-fertilization.--To determine
whether Galax is self-compatible and whether the degree of
self-compatibility differs between cytotypes, we self-pollinated flowers
on six plants in single cytotype populations and three diploid and three
tetraploid plants in mixed cytotype populations. We enclosed a
haphazardly-selected inflorescence on each plant before flowers opened
using mesh bagging. Bagging was applied loosely to allow flowers to
open, mature, and senesce naturally while excluding potential
pollination vectors. The relatively small pore size (~150 [micro]m)
should have eliminated the possibility of wind pollination via pollen
originating on other plants (cf., Berry and Gorchov, 2004). When flowers
on the enclosed inflorescence were open, we briefly removed the bagging
and self-pollinated two flowers by hand with pollen obtained from other
flowers on the same plant. Two to four anthers from each donor flower
were used so that the stigmatic surfaces of recipient flowers were
saturated with pollen. We then applied a small amount of nontoxic fabric
paint to the pedicel of pollinated flowers to allow for the
identification of resulting fruit and reapplied the bagging to prevent
pollen contamination from other plants. Pollinated flowers were
generally near the bottom and among the first to open on a given
inflorescence. Hand-pollinated fruits were collected 8-9 wk later and
their seeds were counted and recorded.

To estimate seed production as a product of autonomous
self-fertilization and to determine whether this differs between
cytotypes, we applied loose-fitting mesh bagging to an entire
inflorescence on six plants in each population before flowers opened. In
mixed-ploidy populations, three diploid and three tetraploid plants were
used. At the end of the season (8-9 wk after the first flowers opened)
the mesh bagging was removed, fruits were collected, and seed number per
fruit was assessed.

Seed production from hand outcrosses and natural pollination.--To
estimate the maximum number of seeds that could be produced per flower
under outcross pollination conditions, six unopened flowers on each of
six plants per population were emasculated while in bud. In mixed-ploidy
populations, three diploid and three tetraploid plants were used. On a
given plant, two buds were near the top, two near the center, and two
near the bottom of a haphazardly-chosen inflorescence. Small mesh bags
were then applied to individual flowers to eliminate the possibility of
natural cross-pollination via insects or wind. Because Galax flowers are
so small with very little space between flowers on an inflorescence, and
because we did not know whether the species was self-compatible, we
removed flowers directly adjacent to the emasculated flowers by cutting
through their pedicels with a scalpel. When stigmas matured (generally
within 2-3 d) we briefly removed the mesh bagging around a given flower
and saturated the stigmatic surface with equal amounts of pollen
obtained from two other individuals from the same population (two
anthers per donor). Using mixedpollen loads helped to ensure seed
production even if one of the donors shared a self-incompatibility
allele with the pollen recipient. Pedicels were labelled with nontoxic
fabric paint and the mesh bagging was put back into place.
Hand-pollinated fruits were collected 8-9 wk later and their seeds were
counted and recorded.

We estimated natural levels of seed production on six plants in
each population at the end of the season by counting the number of
fruits produced on a haphazardly-chosen inflorescence on each plant and
the number of seeds in six fruits, two near the top, two near the
center, and two near the bottom of the inflorescence. In mixed-ploidy
populations, three diploid and three tetraploid plants were used. Seed
production per inflorescence for a plant was estimated as the product of
the mean number of seeds per fruit and the number of fruits per
inflorescence. Aborted fruits were not counted.

Seed number data were analyzed using a three-way ANOVA with
population, pollination type (hand outcrossed vs natural), and flower
location (bottom, middle, and top of inflorescence) as fixed effects.
Plant number was included as a random effect to account for multiple
inflorescence locations sampled on each. Fruit production and total seed
number (natural-log transformed) were analyzed using ANOVA with
population as the independent variable. In both ANOVAs, a priori
contrast was used to compare diploid populations to tetraploid
populations.

RESULTS

Flowering phenology and number.--For almost all plants, regardless
of population or ploidy, flowering began the first week of June and
continued for 6 wk through midjuly (Fig. 1). Although the duration of
flowering did not differ between cytotypes, tetraploids generally
produced flowers earlier than diploids (peak flower production week for
diploids 4.4 [+ or -] 0.07 and tetraploids 4.2 [+ or -] 0.04;
[F.sub.1,28] = 5.17; P = 0.03). Differences between the cytotypes were
greatest at the peak of flower production and the weeks leading up to it
(Fig. 1). Tetraploids had 36% more open flowers than diploids over the
season (tetraploids 331.1 [+ or -] 32.5 and diploids 243.2 [+ or -]
26.6; [F.sub.1,28] = 4.32, P < 0.05).

Floral visitor observations.--We observed 171 floral visitors
representing two taxonomic orders: Hymenoptera and Diptera. The majority
of Hymenoptera visitors were either bumblebees (Bombus spp.), honey bees
(Apis mellifera), or sweat bees (Lasioglossum spp.), while the majority
of Diptera visitors were either hover flies (Taxorrwrus geminatus) or
tachinid flies (Juriniopsis spp.). Hymenoptera were the most common
visitor (65% of visits) with an average of 1.67 [+ or -] 0.097/h and
spent the most time interacting with flowers x= 11.9 [+ or -] 0.7 s)
(Table 2). Diptera comprised 35% of visitors (0.89 [+ or -] 0.069/h),
however their visits were relatively brief x= 5.9 [+ or -] 0.5 s).
Diploids were visited 16% more often than tetraploids (diploid: 1.36 [+
or -] 0.10; tetraploid 1.19 [+ or -] 0.09), but there was no difference
between cytotypes in terms of their visitor fauna or the duration of
visits (Table 2).

Self-compatibility and autonomous self-fertilization.--Seventy of
the 72 self-pollinated flowers did not produce seed and none of the
flowers in bagged inflorescences set seed. Of the two self-pollinated
flowers that did make seeds one was located near the bottom of an
inflorescence on a diploid plant in a diploid population (six seeds);
the other was located near the bottom of an inflorescence on a
tetraploid plant in a mixed-cytotype population (10 seeds). These seed
numbers are small for fruits located near the bottoms of inflorescences
x (22 seeds). Due to low frequency of fruit production and small seed
number, we suspect the seeds produced by these two flowers may have been
a result of pollen contamination and not self-fertilization.

Seed production from hand outcrosses and natural pollination.--Hand
outcrossed flowers produced 16% more seeds than naturally pollinated
flowers (Table 3). Seed production also varied among flowers located on
different regions of an inflorescence, with flowers located near the
bottom producing 25% more seeds (21.6 [+ or -] 0.8) than flowers located
near the middle (17.2 [+ or -] 0.7), which in turn produced almost twice
as many seeds as flowers located near the top (8.9 [+ or -] 0.6; Table
3). There were no differences in seed number per fruit between diploids
and tetraploids and the increase in seed number with cross pollination
and toward the base of the inflorescence was consistent across diploids
and tetraploids (Table 3).

There were no differences in plant-level measures of performance
between diploids and tetraploids. The mean number of fruits per
inflorescence (F]i28 = 0.04, P = 0.85) and total seed production per
inflorescence ([F.sub.1,28] = 0.07, P = 0.79) did not differ between
cytotypes.

DISCUSSION

Overall there were few differences between diploid and tetraploid
Galax urceolata in terms of reproductive ecology. For example, although
the peak of flower production for tetraploids did precede that of
diploids, the overall distributions of flowering times were similar,
resulting in substantial overlap between cytotypes in terms of floral
phenology. In addition, although diploids were visited more frequendy by
pollinators than tetraploids, we found no evidence for differences
between cytotypes in terms of the taxonomic composition of their
pollinator communities. Moreover, both diploid and tetraploid Galax
appear to be self-incompatible. Finally, fruit production is comparable
across cytotypes, with both having increased seed production in
hand-outcrossed flowers suggesting pollen limitation. In total, while
there are significant differences in some aspects of the reproductive
ecology between diploid and tetraploid Galax, the overall pattern is one
of similarity.

Our study indicates Galax is self-incompatible and insect
pollinated. Hymenoptera were twice as common as Diptera and interacted
with flowers for twice as long. Therefore, it is likely that
Hymenoptera--most notably a combination of bumble bees, honey bees and
sweat bees--are the most important group of pollinators for Galax,
facilitating the majority of the species' pollen transfer. Plants
set no fruit by autonomous pollination and virtually no fruit by hand
self-pollination, indicating that Galax is self-incompatible and relies
on insects for between genet pollen movement to produce seeds.
Self-incompatibility in both diploid and tetraploid Galax is somewhat
unexpected in that polyploidy is often associated with a breakdown in
self-incompatibility systems (cf., Miller and Venable, 2000; Robertson
et al., 2010; but see Mable, 2004) and polyploid angiosperms often
exhibit higher levels of self-fertilization than their diploid relatives
(Barringer, 2007).

Our data suggest diploid and tetraploid Galax are both pollen
limited. Natural levels of seed production ranged widely, though this
variation was not related to cytotype. Hand-pollinated flowers produced
on average 16% more seed than open-pollinated flowers, suggesting pollen
limitation reduces seed production in natural populations of Galax
(whether in terms of total pollen or nonself pollen received). However,
the degree of pollen limitation did not depend on cytotype, and there
were no differences between cytotypes in terms of the average numbers of
seeds per fruit or the numbers of fruits per inflorescence.
Interestingly, the occurrence of pollen limitation in Galax suggests
pollen competition for access to ovules is limited, which in turn could
facilitate the success of between-cytotype seed production even if
intercytotype fertilization is infrequent or inefficient.

Intercytotype crosses are expected to produce offspring with low
fitness. For example diploid-tetraploid crosses often produce sterile
triploids (Levin, 1975; Ramsey and Schemske, 1998; Sutherland and
Galloway, 2016). Therefore, in populations in which both diploid and
tetraploid cytotypes occur, or in areas in which diploid and tetraploid
populations are in relatively close proximity, selection to reduce the
frequency of intercytotype crosses may result in character displacement
for traits that influence the likelihood such crosses occur, i.e.,
prezygotic isolation. Our study found few differences between
populations of diploid and tetraploid Galax in terms of their
reproductive ecology, a finding that can simultaneously help to explain
the lack of genetic divergence between cytotypes and the relatively
frequent formation of triploid lineages (at least 31 times) and
tetraploid lineages (at least 46 times) within the species (Servick et
al., 2015). Indeed, the prevalence of triploids and the recurrent
formation of both triploid and tetraploid lineages suggests tetraploidy
in Galax might commonly arise via a so-called triploid bridge (Harlan
and de Wet, 1975; Ramsey and Schemske, 1998), as this process would be
facilitated by overlapping flowering phenologies, shared pollinators,
and self-incompatibility. The performance of intercytotype crosses has
not been investigated and would illuminate this possibility.

Acknowledgments.--The authors thank The Highlands Biological
Station and the William Chambers Coker Fellowship in Botanical Research
for their support. Thanks also to Brett Jones for assistance in the
field.